【作者】 高岩；

【导师】 杨贞耐；

【作者基本信息】 吉林农业大学 ， 发酵工程， 2012， 硕士

【摘要】 凝乳酶是干酪生产中促使牛乳凝固的关键性酶,对于干酪的质构及风味的形成有非常重要的作用。凝乳酶在食品、医疗、工业等行业也都有广泛应用,是重要的工业酶之一。按其来源分为动物凝乳酶、植物凝乳酶、微生物凝乳酶以及基因工程凝乳酶,而微生物来源由于具有产酶周期短、生产成本低等特点,已经成为最有前途的凝乳酶来源。本论文以枯草芽孢杆菌JAASB为出发菌株,通过多种诱变方法筛选出一株较为理想的菌株JAASB-14,并采用液态发酵技术,对其最佳发酵培养基、最佳发酵条件以及其酶学性质进行了研究,最后模拟工业生产对菌株进行50L发酵罐的扩大培养,并用获得的凝乳酶制了切达干酪,分析了切达干酪的理化性质。主要研究结果如下：1.从实验室保藏的20株枯草芽孢杆菌中筛选出三株产凝乳酶的菌株,通过产酶曲线选出其中较理想的菌株JAASB作为出发菌株,在酪蛋白平板上以凝乳圈直径与菌落直径的比值为指标进行初筛,在液态发酵培养基上以凝乳酶活力为指标进行复筛,通过紫外诱变与DES的复合诱变,从中选育到三株具有遗传稳定性的高产菌株,即JAASB-3、JAASB-5、JAASB-14其凝乳活力分1142.6SU/mL、1263.2SU/mL.1333.3SU/mL,比出发菌株分别提高了52%、68%、78%。2.在基础培养基的基础上,通过单因素试验和响应面分析法确定了最适培养基为玉米粉1.5%、豆粨粉2.0%、NaCl为0.6%、MgSO4·7H2O0.6%、Na2HPO4·12H2O0.4%。通过单因素试验探讨了各种培养条件对凝乳酶活力的影响。确定了摇床培养最佳发酵条件为最适起始pH值自然(pH值为7.4-7.6),接菌量为5%,摇床转速为280r/min,培养时间为24h。3.对枯草芽孢杆菌凝乳酶的酶学性质进行了研究并与商品凝乳酶作了对比。枯草芽孢杆菌凝乳酶的最适反应温度为55℃,最适pH值为5.0,在pH值为6.0时最稳定,在65℃保温60min酶活基本丧失,Mn2+、Li+、Fe2+、Na+、K+、Mg2+、Ca2+对该凝乳酶有促进作用,Mg2+、Ca2+促进作用较为明显,而Cu2+对该酶有明显的抑制作用；脱脂乳浓度为10%时,凝乳酶活力最大；酶修饰剂中β-巯基乙醇对凝乳酶的活力影响较小,EDTA可抑制该凝乳酶的活力；蛋白酶抑制剂α-phenantroline可以明显抑制酶活性,证实该酶为金属蛋白酶类。在与商品酶对比中发现枯草芽孢杆菌凝乳酶与商品酶的酶学性质相近。4.利用50L发酵罐进行放大培养,按照最佳的培养基进行发酵,18h发酵液的凝乳活力能到达2667SU/mL,利用发酵罐发酵后的凝乳酶制作成切达干酪,并与商品凝乳酶制作的切达干酪进行对比,测定不同时期干酪的理化指标、可溶性氮和干酪质构,其结果显示,添加枯草芽孢杆菌凝乳酶制作的干酪与商品酶对照组没有明显差别。感官评定结果显示,使用枯草芽孢杆菌凝乳酶的干酪,在风味上稍逊于使用商品凝乳酶的干酪。更多还原

【Abstract】 Chymosin is an important enzyme which made the milk coagulation in the production of cheese,and it has an important role for the form of cheese texture and flavor. Chymosin is an important enzyme, one of the industrial enzymes widely applied in food,medicine industry and other field.According to different sources,the rennet can be divided into four categories:rennet enzume from animal,plant,microbe and genetic engineering.Because of the merits,such as wide source, short production period, and low cost,microbe has become the most promising source of chymosin.The research taked Bacillus subtilis JAASB as the initial strain.throuth some mutation methods obtained a bacteria named JAASB-14, then fermented with liquid fermentation.This paper studied the condition of enzyme-producing, the optimum medium and enzyme properties.At last using50L scale-up fermentation which simulting the industrial conditions,and made chedder cheese by milk clotting enzyme which produced by Bacillus subtilis,and studied the composition of chedder cheese.The main contents of the study were as follows:l.The research obtained3strains of bacteria from20strains of bacteria which conserved from a lab.Through the curve of chymosin obtained a ideal bacteria,this bacteria as the initial strain. Effect of mutation was determined by preliminary screening based on the ratio between milk-clotting zone and colony diameter, and secondary screening based on liquid fermentation. The starting strain Bacillus subtilis JAASB producing chymosin was mutated by UV and DES, three mutant strains with good genetic stability and high chymosin activity were obtained. The chymosin activity produced by JAASB-3JAASB-5and JAASB-14was1142.6SU/mL、1263.2SU/mL、1333.3SU/mL, respectively.Comparing with the starting strain, chymosin activity produced by JAASB-3JAASB-5and JAASB-14was increased by52%、68%and78%,respectively.2.Based on the basic medium, Through the single factor experiment and the response surface method the research got the most suitable medium:corn starch1.5%,beans power2.0%,、NaCl0.6%、MgSO4·7H2O0.5%、 Na2HPO4·12H2O0.4%。. Through the single factor experiment study of the culture conditions and medium of nutrition conditions.The optimum culture condition text showed:the initial pH natual(The culture medium is neutral), the amount of inoculation is5%, rate of rotation280r/min.incubation time is24h.3.Characterization of chymosin produced by Bacillus subtilis was studied.The results showed that the optimum temperature of chymosin was55℃, and the optimum coagulating pH was5.0.The clotting activity was more stable at pH6.0. The enzyme almost lost activity by retaining after60min at65℃. The metal ions Mn2+、Li+、Fe2+、Na+、K+、Mg2+、Ca2+increased the clotting activity and Mg2+、Ca2+showed the greatest effect, while Cu2+inhibited the clotting activity. As enzyme modifiers, β-mercaptoethanol had no clear effect on milk-clotting activity, while EDTA inhibited the clotting activity. As protein inhibitors, σ-phenantroline strongly inhibited the clotting activity, so the enzyme is a metalloproteinase. Experimental results showed that chymosin produced by Bacillus subtilis had the similar characteristics to the commercial chymosin.4. The research used50L scale-up fermentation which using the optimized medium,after18h the clotting activity reached2677SU/m L.Cheddar cheese was maded using chymosin produced by Bacillus subtilis,and it was compared with commercial chymosin cheese. In the test the changese of diferent period of physicochemical indexes, water-soluble nitrogen and texture were determined.The results showed that there is no difference between cheddar cheese made by rennet products by Bacillus subtilis and commercial chymosin.The results of sensory evaluation show that cheddar cheese made by rennet products by Bacillus subtilis is less then commercial chymosin in the flavor.更多还原